Tsipora Malachi

Platelet Count and Thrombopoietic Activity in Patients with Chronic Renal Failure

The frequency of thrombocytopenia in patients with chronic renal failure (CRF) is controversial. This study was undertaken to investigate the platelet count in 55 patients with end-stage renal disease on maintenance hemodialysis and in 19 patients with CRF before hemodialysis had begun. In both groups platelet counts were similar and significantly reduced, 175,000 +/- 6,500 and 181,000 +/- 10,800 compared to 253,000 +/- 3,700/mm3 in the control (p less than 0.0001). 31% of hemodialysis patients had thrombocytopenia (platelet count less than 150,000/mm3). The megakaryocyte number in their bone marrow aspirate was not reduced. Primary renal disease, androgen treatment or parathyroidectomy did not affect the platelet count. Thrombopoietic activity using 75Se-selenomethionine incorporation into platelets measured in 7 thrombocytopenic patients was found to be reduced, 6.77 +/- 0.29 vs. 9.06 +/- 0.27 (X 10(-2)%: p less than 0.001). This study shows that the platelet count is reduced and mild thrombocytopenia is frequent in patients with CRF. A possible cause for the platelet count reduction is insufficient thrombopoietic activity.

H2 O2 induces DNA repair in mononuclear cells: Evidence for association with cytosolic Ca2+ fluxes

Cellular DNA repair systems are induced whenever DNA is damaged. Reactive oxygen species (ROS) are generated, in vivo, in the tissues as a result of regular cellular metabolism or after exposure to oxidizing agents, such as ultraviolet (UV) irradiation. It has been suggested that ROS mediate DNA damage. The objectives of the study were as follows: (1) to investigate whether hydrogen peroxide (H2O2), the commonly occurring cellular ROS, induces DNA repair as a response to the damage it probably causes; (2) to evaluate whether H2O2-induced DNA repair, if present, is signaled through a Ca2(+)-dependent pathway via the tyrosine kinase signal transduction. H2O2 was found to induce DNA repair in human peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. The recovery of RNA synthesis, which occurred after DNA repair, confirmed that transcribable DNA was repaired. The inhibition of tyrosine kinase activity by genistein reduced the DNA repair significantly. Furthermore, H2O2 caused a dose-dependent significant rise in cytosolic calcium ((Ca2+)i). H2O2 also induced a small rise in (Ca2+)i of cytosolic Ca2(+)-depleted cells, probably reflecting the release of Ca2+ from internal stores. Genistein inhibited both Ca2+ influx and Ca2+ release from internal stores. In summary, H2O2 induced a DNA repair synthesis that was in part Ca2+ dependent and signaled via tyrosine kinase. The changes in DNA repair paralleled changes in (Ca2+)i. The H2O2-induced (Ca2+)i rise was mostly the result of influx, but to some degree it was also due to the translocation of Ca2+ from internal stores.

Parathyroid hormone effect on the fragility of human young and old red blood cells in uremia.

Parathyroid hormone (PTH) is elevated in patients with chronic renal failure (CRF) and was suggested to be one of the factors responsible for the anemic syndrome of these patients because it raises the osmotic fragility of the red blood cells (RBC). In the present study, the youngest and oldest RBC were separated from circulating erythrocytes by high-speed centrifugation. The age distribution was described, and the effect of PTH on the different age groups was investigated. Median density (MD) and glutamic-oxaloacetic transaminase (GOT) activity were chosen as age markers. MD (1.0985 +/- 0.00087) and GOT activity (12.49 +/- 2.083 IU/g Hb) of the young uremic cells did not differ significantly from the values of young normal cells (1.0987 +/- 0.00046 and 10.36 +/- 1.174 IU/g Hb, respectively). The MD of the oldest cells, however, was lower (1.1048 +/- 0.00054) and GOT was higher (6.60 +/- 1.1019 IU/g Hb) in the uremic than in the control cells (1.1093 +/- 0.00175 and 3.77 +/- 0.233 IU/g Hb, respectively). These results indicate that the life span of RBC in uremics is shorter than normal and that an enrichment of circulating RBC by young cells occurs in uremic patients. The median osmotic fragility (MOF) of the young cells was lower in both uremic (0.376 +/- 0.006) and control patients (0.378 +/- 0.003) than the MOF of old cells (0.402 +/- 0.005 and 0.392 +/- 0.004, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of immunosuppressive drugs on spontaneous DNA repair in human peripheral blood mononuclear cells.

INTRODUCTION Immunosuppressive treatment increases the risk of post-transplant cancer. Cyclosporine reduced UV-induced DNA repair by peripheral blood mononuclear cells (PBMC) and increased cancer incidence in kidney transplant recipients. Calcineurin inhibitors (CNI), but not mammalian target of rapamycin (mTOR) inhibitors or mycophenolic acid, suppressed H₂O₂-induced DNA repair in human peripheral blood mononuclear cells (PBMC) in vitro at maintenance drug concentrations. DNA repair, when measured in quiescent cells, is named spontaneous DNA repair, and represents a basal ongoing DNA repair in response to endogenous DNA damage. The effect of immunosuppressive drugs on spontaneous DNA repair has not been investigated. AIM To investigate the effect of currently used immunosuppressive drugs on spontaneous DNA repair. METHODS Spontaneous DNA repair by human PBMC was tested in vitro in the presence of the CNI-cyclosporine and tacrolimus; mycophenolic acid (MPA); and the mTOR inhibitors-sirolimus and everolimus, at low to high nontoxic concentrations. RESULTS Cyclosporine and tacrolimus suppressed spontaneous DNA repair throughout the tested dose range. In contrast, MPA, sirolimus and everolimus did so only at the high doses. CONCLUSION A reduction in CNI dosage may lead to a decrease in the occurrence of post-transplant malignancy.

Spontaneous DNA Repair Increases during Hemodialysis

Background: Hemodialysis (HD) patients are subjected to increased oxidative stress. Oxidative stress causes DNA damage, which may be repaired by a DNA repair system. ‘Spontaneous DNA repair’ expresses DNA repair of in vitro unstimulated cells. The aim of the study was to evaluate the effect of one HD session on spontaneous DNA repair in peripheral blood mononuclear cells (PBMC). Methods: PBMC were separated from blood samples for the determination of spontaneous DNA repair, measured by 3H-thymidine incorporation, before and immediately after one HD session. Percent double-stranded DNA (ds-DNA) was measured by the fluorometric assay of DNA unwinding (FADU). Results: DNA repair increased significantly following HD. To examine if this increase was caused by newly produced DNA damage, we studied the effect of HD on percent ds-DNA in PBMC. HD significantly reduced percent ds-DNA, indicating increased DNA breakage. By repeating FADU in the presence of formamidopyrimidine-DNA glycosylase (Fpg), which nicks DNA at oxidized purine sites, we could show that the increased DNA damage was caused by oxidation. Conclusion: Spontaneous DNA repair increases during HD in response to an increase in DNA damage induced by oxidative stress.

Effect of immunosuppressive drugs on DNA repair in human peripheral blood mononuclear cells

INTRODUCTION Cancer is a major cause of mortality among transplant recipients. Immunosuppressive treatment is a modifiable factor contributing to this phenomenon. Cyclosporine in kidney transplant recipients was associated with reduced UV-induced DNA repair by peripheral blood mononuclear cells (PBMC) and increased cancer rate. H(2)O(2) is a common cellular reactive oxygen species (ROS), which induces DNA damage followed by DNA repair. AIM To investigate the effect of currently used immunosuppressive drugs on DNA repair. METHODS H(2)O(2)-induced DNA repair by human PBMC was tested in vitro in the presence of the calcineurin inhibitors (CNI) cyclosporine and tacrolimus, mycophenolic acid (MPA), and the mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus, at low to high non-toxic concentrations. The effect of combination therapy at maintenance levels was also tested. RESULTS Cyclosporine and tacrolimus suppressed DNA repair throughout the tested dose range. In contrast, MPA, sirolimus and everolimus did so only at the high doses. Maintenance doses of a combination of tacrolimus and MPA, the most frequent treatment regimen, reduced DNA repair, while MPA with sirolimus or everolimus did not. CONCLUSION In an attempt to reduce the risk of post-transplantation malignancy, treatment protocols may be modified by reducing CNI dose.

Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin

Background DNA repair is a cellular defence mechanism responding to DNA damage caused in large part by oxidative stress. There is a controversy with regard to the effect of red blood cells on DNA damage and cellular response. Aim To investigate the effect of red blood cells on H2O2-induced DNA damage and repair in human peripheral blood mononuclear cells. Methods DNA breaks were induced in peripheral blood mononuclear cells by H2O2 in the absence or presence of red blood cells, red blood cells hemolysate or hemoglobin. DNA repair was measured by 3H-thymidine uptake, % double-stranded DNA was measured by fluorometric assay of DNA unwinding. DNA damage was measured by the comet assay and by the detection of histone H2AX phosphorylation. Results Red blood cells and red blood cells hemolysate reduced DNA repair in a dose-dependent manner. Red blood cells hemolysate reduced % double-stranded DNA, DNA damage and phosphorylation of histone H2AX. Hemoglobin had the same effect as red blood cells hemolysate on % double-stranded DNA. Conclusion Red blood cells, via red blood cells hemolysate and hemoglobin, reduced the effect of oxidative stress on peripheral blood mononuclear cell DNA damage and phosphorylation of histone H2AX. Consequently, recruitment of DNA repair proteins diminished with reduction of DNA repair. This suggests that anemia predisposes to increased oxidative stress induced DNA damage, while a higher hemoglobin level provides protection against oxidative-stress-induced DNA damage.

Lymphocytic intracellular calcium in a patient with complicated verapamil overdose.

Overdose with calcium channel blockers (CCBs) may lead to serious complications. CCBs act by blocking calcium entry into the cell, thus lowering intracellular calcium ([Ca2+]i). [Ca2+]i during CCB overdose has not yet been reported. We measured [Ca2+]i in lymphocytes of a patient with acute verapamil overdose with a complex clinical picture. A 59-year-old woman was admitted after a suicidal ingestion of 7200 mg of a sustained-release verapamil preparation. She presented with hypotension, complete atrioventricular block, stupor, hypokalemia, and hyperglycemia. Acute oliguric renal failure, acute pancreatitis, and the adult respiratory distress syndrome further complicated her medical course. Treatment was supportive and she recovered completely. Intracellular calcium ([Ca2+]i) was measured in the patients lymphocytes using a spectrofluorometer with the calcium-sensitive dye Fura-2-acetoxymethyl ester. Thirty nine hours after the ingestion, [Ca2+]i was low at 52 nM (compared with 80 nM in a healthy control subject). Lymphocytic [Ca2+]i did not respond to stimulation with phytohemagglutinin (PHA). Fourteen days after the verapamil overdose, after the patient had recovered completely, lymphocytic [Ca2+]i was still low at 55 nM. At this time, there was an incomplete response to PHA in the lymphocytes. Three months after the ingestion, [Ca2+]i was normal, with a normal response to PHA. Verapamil overdose may run a complex clinical course, but full recovery is to be hoped for with full supportive care. Cellular intoxication, as reflected by low lymphocytic [Ca2+]i, is prolonged and lags behind the clinical recovery by weeks.

Inhibition of mitochondrial function reduces DNA repair in human mononuclear cells

BACKGROUND Mitochondria provide ATP and Ca(2+) needed for DNA repair, but also produce reactive oxygen species (ROS), which may damage DNA. AIM To investigate the effect of mitochondrial function inhibition on DNA repair. METHOD Five mitochondrial inhibitors acting at various sites of electron transport were studied. Human peripheral blood mononuclear cells, spontaneous and H(2)O(2)-induced DNA repair, as well as %-double-stranded-DNA, were measured. RESULTS All mitochondrial inhibitors suppressed spontaneous and H(2)O(2)-induced DNA repair. However, their effect on %-double-stranded-DNA differed, which is partly related to ROS suppression. CONCLUSION Mitochondrial inhibition may enhance efficacy and reduce toxicity of radiation and cytotoxic drugs therapy.

Biochemical changes associated with the osmotic fragility of young and mature erythrocytes caused by parathyroid hormone in relation to the uremic syndrome.

The effect of parathyroid hormone at concentrations found in uremic patients on erythrocytes (RBC) from newborn and adult rabbits was studied in relation to the fragility pattern in hypotonic salt solutions and the activities of Ca- and Mg-dependent ATPases. Median osmotic fragility of RBC from newborn rabbits was significantly lower than in mature rabbits. Parathyroid hormone (PTH) stimulated to a greater extent the mean osmotic fragility in RBC from newborn rabbits, than in those from adults. Similarly, the hormone stimulated to a much greater extent the Ca-ATPase but not the Mg-ATPase in RBC from the newborn rabbits, in comparison to those from adult rabbits. PTH, which is greatly elevated in the blood of patients with chronic renal failure, may be one cause of the anemia seen in these patients, and its effect, which is mediated by Ca-ATPase activity, is stronger on young RBC. There were significant morphological changes in the young RBC caused by PTH, as seen with scanning electron microscopy.